Martine Besouw

Stem cell microvesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro.

Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100–400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNSRed transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.

Fertility status in male cystinosis patients treated with cysteamine.

OBJECTIVE To analyze the fertility status in adult, male cystinosis patients treated with cysteamine. Cystinosis is an autosomal recessive disease leading to intralysosomal cystine accumulation. Worldwide, a few female cystinosis patients have given birth. However, no male cystinosis patients are known to have induced pregnancy. Adequate cysteamine treatment might improve male fertility. PATIENT(S) Seven male cystinosis patients (19-43 years) were submitted. INTERVENTION(S) Glomerular filtration rate was estimated using the Cockcroft formula. Serum LH, FSH, testosterone, and inhibin B were determined. Semen analysis was performed in five patients. Testicular biopsy was performed in one patient. RESULTS Glomerular filtration rate ranged between 10 and 110 (normal >90) mL/min/1.73 m(2), LH and FSH levels ranged between 7.4 and 235.0 (normal 1.4-8.5) E/L and 6.8-298.0 (normal 1.5-11) E/L, respectively. Plasma testosterone level ranged between 8.7 and 31.3 (normal 11-45) nmol/L; plasma inhibin B level ranged between 10 and 210 (normal 150-400) ng/L. All of the collected sperm samples showed azoospermia. The testicular biopsy showed a Johnson score of 8 to 9. CONCLUSION(S) We demonstrate azoospermia in male cystinosis patients, even if adequately treated with cysteamine starting from an early age. The finding of spermatogenesis in the testis biopsy of one patient may provide opportunities to male cystinosis patients to produce their own offspring by in vitro fertilization after testicular sperm extraction.

Copper deficiency in patients with cystinosis with cysteamine toxicity

OBJECTIVES To assess whether copper deficiency plays a role in the recently described cysteamine toxicity in patients with cystinosis, and to examine whether polymorphisms in copper transporters, lysyl oxidase, and/or type I procollagen genes could be responsible for the occurrence of cysteamine toxicity in a small subset of patients with cystinosis. STUDY DESIGN Thirty-six patients with cystinosis were included: 22 with Fanconi syndrome (including 7 with cysteamine toxicity), 12 after renal transplantation, 1 receiving hemodialysis, and 1 with ocular cystinosis. Serum copper and ceruloplasmin levels and urinary copper/creatinine ratio were measured. Genes ATP7A and CTR1 (encoding copper transporters), LOX (encoding lysyl oxidase), and COL1A1 and COL1A2 (encoding type I procollagen) were analyzed in patients with (n = 6) and without (n = 5) toxicity. Fibroblast (pro)collagen synthesis was compared in patients with (n = 3) and those without (n = 2) cysteamine toxicity. RESULTS All 22 patients with Fanconi syndrome had increased urinary copper excretion. Serum copper and ceruloplasmin levels were decreased in 9 patients, including all 7 patients with cysteamine toxicity. No specific sequence variations were associated with toxicity. All fibroblasts exhibited normal (pro)collagen synthesis. CONCLUSION Patients with cystinosis with cysteamine toxicity demonstrate copper deficiency. This can cause decreased activity of lysyl oxidase, the enzyme that generates the aldehydes required for collagen cross-linking. Thus, copper supplementation might prevent cysteamine toxicity.

Halitosis in cystinosis patients after administration of immediate-release cysteamine bitartrate compared to delayed-release cysteamine bitartrate

Halitosis due to dimethylsulfide (DMS) generation is a major side effect of cysteamine in the treatment of cystinosis. Recently, an enteric coated formulation of cysteamine bitartrate (RP103) administered twice daily was demonstrated to be non-inferior for lowering WBC cystine levels compared to the non-enteric coated formulation (Cystagon®), administered 4 times per day. Since both formulations had different pharmacokinetic profiles, we compared DMS breath levels after administration of either RP103 or Cystagon® in four cystinosis patients. Although cysteamine areas under the curve (AUCs) were comparable, AUC of DMS was lower after the administration of RP103 compared to Cystagon®. This observation is of importance in cystinosis patients, since halitosis hampers compliance with cysteamine therapy.

Pharmacokinetics of cysteamine in a cystinosis patient treated with hemodialysis

Sirs, Cystinosis is a rare autosomal recessive disease caused by mutations in the CTNS gene, resulting in lysosomal accumulation of the amino acid cystine in cells throughout the body. Most patients develop general proximal tubular damage, called renal Fanconi syndrome, within the first year of life. Besides kidney involvement, the disease can manifest in the eyes, various endocrine organs, central nervous system, and muscles. If not treated with the cystine-depleting agent cysteamine, patients will develop end-stage renal disease (ESRD) around the age of 10 years. The advised dose of cysteamine is 1.30 g/m 2 body surface area, with a maximum of 1.95 g/m 2 , divided into four equal doses. Recently, questions have been raised whether or not to decrease cysteamine dose in cystinosis patients with ESRD, as clearance of the drug might be lowered. We present a pharmacokinetic study of cysteamine in a cystinosis patient with ESRD treated with hemodialysis. The patient is a 19-year-old man. The diagnosis of cystinosis was made at the age of 1 year and confirmed by mutation analysis of the CTNS gene, revealing a homozygous c.926 duplication. At the time of this study, he had been on hemodialysis since 1 year after failure of his second kidney graft. Cysteamine plasma levels were measured before and every 60 min after ingestion of 15 mg/kg cysteamine for a total duration of 6 hours. This study of cysteamine pharmacokinetics was conducted in fouradditionalcystinosispatients(agerange12–29years), all of whom had a functioning renal graft [glomerular filtration rate (GFR) 72–109 mL/min/1.73 m 2 ]. The results are shown in Fig. 1. A reduced GFR did not lead to increased plasma levels of cysteamine. Conversely, the patient on hemodialysis had the lowest area under the curve (AUC) compared with the patients with a functioning renal graft (46 vs. 58–136 μmol/l, respectively).

Cystinosis: a new perspective

Cystinosis is a rare, autosomal recessive inherited lysosomal storage disease. It is the most frequent and potentially treatable cause of the inherited renal Fanconi syndrome. If left untreated, renal function rapidly deteriorates towards end-stage renal disease by the end of the first decade of life. Due to its rarity and non-specific presentation, the entity is often not promptly recognized resulting in delayed diagnosis. Two major milestones in cystinosis management, cystine-depleting therapy with cysteamine and renal allograft transplantation, have had a considerable impact on the natural history and prognosis of cystinosis patients. However, due to its significant side effects and a strict 6-hourly dosing regimen, non-adherence to the immediate release of cysteamine bitartrate formulation (Cystagon®) is a major issue that might affect long-term outcome. Recently, a new twice-daily administered delayed-release enteric-coated formula of cysteamine bitartrate (ProcysbiTM) has been approved by the European Medical Agency for the treatment of cystinosis, and has been shown to be safe and effective. This delayed-release cysteamine has the potential to improve compliance and hence prognosis, through its better dosing regimen, positive impact on quality of life and possibly less side-effects, and is now tested in an ongoing long-term clinical trial. Longer survival of patients with cystinosis makes transition from pediatric to adult-oriented care another challenge in cystinosis management and requires an extended multidisciplinary approach.

Growth hormone producing prolactinoma in juvenile cystinosis: a simple coincidence?

Juvenile cystinosis was diagnosed in a patient who presented with severe headache attacks and photophobia. Treatment with oral cysteamine and topical cysteamine eye drops was started. One-and-a-half years later, he developed unilateral gynecomastia and elevated prolactin and growth hormone levels. A pituitary macroprolactinoma was discovered and successfully treated with the dopamine agonist cabergoline. Increased serum growth hormone levels were attributed to enhanced growth hormone production by the prolactinoma and somatostatin inhibition by cysteamine. Although the occurrence of prolactinoma in this patient could be a simple coincidence, it might also be a rare yet unrecognised complication of cystinosis.

Improving the prognosis of nephropathic cystinosis

Cystinosis is an autosomal recessive inherited lysosomal storage disease. It is characterized by generalized proximal tubular dysfunction known as renal Fanconi syndrome and causes end-stage renal disease by the age of about 10 years if left untreated. Extrarenal organs are also affected, including the thyroid gland, gonads, pancreas, liver, muscle, and brain. Treatment consists of administration of cysteamine, resulting in depletion of cystine that is trapped inside the lysosomes. Since cysteamine has a short half-life, it should be administered every 6 hours. Recently, a new delayed-release formulation was marketed, that should be administered every 12 hours. The first studies comparing both cysteamine formulations show comparable results regarding white blood cell cystine depletion (which serves as a measure for cystine accumulation in the body), while a slightly lower daily dose of cysteamine can be used.

Eight years experience with in-center nocturnal hemodialysis

Abstracts 48th ESPN Meeting, Brussels, September 2015s 48th ESPN Meeting, Brussels, September 2015 O 01 CAN EARLY RECOGNITION OFAKI IN CHILDREN BE ACHIEVED BY USING AN ALGORITHM (PRELIMINARY RESULTS): ON BEHALF OF BRITISH ASSOCIATION FOR PAEDIATRIC NEPHROLOGY Jelena Stojanovic, Nabil Melhem, Sheetal Bhojani, Manish D Sinha, David Milford Evelina London Childrens Hospital, London, UK; Royal Hospital for Sick Children, Glasgow, Scotland; Birmingham Childrens Hospital, Birmingham, UK Introduction: The aim of the study was to validate recently proposed algorithm ‘Standardising early identification of Acute Kidney Injury’ introduced byNHSEngland in a paediatric setting and to investigate recognition and management of AKI. This multi-centre national project was supported by UK Renal Registry and British Association for Paediatric Nephrology. Material and methods: In part one of the audit, all creatinine measurements performed at each of six centres over a six month period were evaluated electronically using the algorithm. In part two, 180 children from six centres were randomly selected and their case notes reviewed. Here, we report preliminary results on data analysed from two tertiary children’s and one district general hospital in the UK. Information was obtained from paper and electronic patient’s notes. AKI stage 1 is a rise of >1.5x baseline creatinine level; AKI stage 2 is a rise of>2x baseline and AKI stage 3 is a rise of>3x baseline. Results: 33,663 creatinine measurements were analysed during the study period using the AKI algorithm.We identified 1,940 AKI 1 episodes (604 children), 479 AKI 2 (158 children) and 756 AKI 3 (112 children). Overall 666 unique children had one or more AKI episodes.We reviewed case notes of 66 children (39 boys) age range 28 days to 17 years. On clinical review of case notes, AKI was recognised in 18 patients (27.3%) only. Of all patients, 17% had prexisting renal condition. 94% children had a follow up arranged with creatinine normalising in 75% of those tested. A third of patients had urine tested and two thirds had medication dosage adjusted to estimated GFR. Conclusions: The proposed algorithm provides an electronic means of identifying children with AKI and highlighting its severity. Our preliminary data suggest that AKI remains clinically under recognised in clinical settings. Timely recognition and optimal management of AKI is important to improve longer term renal outcomes. O 02 EPIGENETIC REGULATION BY HDAC PROTEINS PLAYSACRITICALROLE INTHEPROGRESSIONOFRENAL FIBROSIS Scott Manson, Qiusha Guo, Katelynn Moore, Paul Austin Washington University, Washington, The United States of America Introduction: Chronic kidney disease is associated with changes in the expression of approximately 10% of the genome. The histone deacetylases (HDACs) are a family of 10 related proteins which are among the most widely expressed and crucial regulators of gene transcription. In this study, we examine the biologic and therapeutic importance of HDAC proteins during disease progression. Material and methods: Chronic renal injury was modeled in vivo in mice by unilateral ureteral obstruction (UUO). The role of HDAC proteins was assessed by using a variety of molecular techniques and treatment with the broad spectrumHDAC inhibitor Trichostatin A (TSA). Results:UUO leads to a dramatic increase in the protein levels of 9 of the 10 HDAC isoforms. Notably, there is a 6.1-fold increase in HDAC8 expression that localizes specifically to pericyte-derived myofibroblasts, the cell population which accounts for the majority of matrix production during renal fibrosis. To better understand the importance of these findings, we treated mice with the HDAC inhibitor TSA. This resulted in a 3.4-fold increase in the anti-fibrotic gene BMP7, a 41.6% decrease in the matrix protein COLIA1, and a 61.6% decrease in the myofibroblast differentiation marker α-SMA following UUO. These changes in gene expression culminate in a 77.9% decrease in the interstitial proliferative response, a 43.0% decrease in myofibroblast number, 31.1% decrease in renal fibrosis, 42.8% decrease in apoptosis, and a 43.4% decrease in the loss of renal architecture. [All results are p<0.05] Conclusions: Chronic renal injury is associated with a dramatic increase in HDAC protein levels that stimulates pro-fibrotic gene expression and suppresses anti-fibrotic gene expression. Importantly, treatment with HDAC inhibitors reverses these changes in gene expression and inhibits the development of renal fibrosis. This suggests that HDAC inhibitors may serve as effective therapies to inhibit disease progression. O 03 ECULIZUMAB TREATMENT IN SEVERE PEDIATRIC STEC-HUS, A MULTICENTRIC RETROSPECTIVE STUDY Percheron Lucas, Gramada Raluca, Decramer Stephane, Harambat Jerome, Eckart Philippe, Bourdat-michel Guylhene, Leroy Valerie, Sellier-leclerc Anne-laure, Adra Anne-laure, Allain-launay Emma, Berard Etienne, Bouchireb Karim, Fila Marc, Pietrement Christine, Merieau Elodie, Lapeyraque Anne-laure, Chehade Hassid, Fremeaux-bacchi Veronique, Dimeglio Chloe, Garnier Arnaud Service De Nephrologie Medecine Interne, Hopital Des Enfants, Chu Purpan, Toulouse, France; Service De Neuroradiologie Diagnostique Et Thérapeutique, Chu Purpan, Toulouse, France; Service De Nephrologie Pediatrique, Hopital Pellegrin-enfants, Chu Bordeaux, Bordeaux, France; Service De Pediatrie Medicale, Hopital Cote De Nacre, Chu Caen, Caen, France; Service De Pediatrie, Hopital Couple-enfants, Chu Grenoble, Grenoble, France; Service De Nephrologie Pediatrique, Hopital Jeanne De Flandre, Chu Lille, Lille, France; Service De Nephrologie Pediatrique, Hopital Femme Mere Enfant, Hospices Civils De Lyon, Lyon, France; Service De Nephrologie Pediatrique, Hopital Arnaud De Villeneuve, Chu Montpellier, Montpellier, France; Service De Nephrologie Pediatrique, Hopital Mere-enfants, Chu Nantes, Nantes, France; Service De Nephrologie Pediatrique, Hopital Archet 2, Chu Nice, Nice, France; Service De Nephrologie Pediatrique, Hopital Necker Enfants Malades, Assistance Publique-hopitaux De Paris, Paris, France; Service De Nephrologie Pediatrique, Hopital Robert Debre-paris, Assistance Publique-hopitaux De Paris, Paris, France; Service De Pediatrie, Hopital Americain, Chu Reims, Reims, France; Service De Nephrologie, Hopital Clocheville, Chu Tours, Tours, France; Service De Nephrologie Pediatrique, Chu De Sainte-justine, Montreal, Canada; Service De Nephrologie Pediatrique, Chu De Lausanne, Lausanne, Switzerland; Laboratoire D’immunologie, Hopital Europeen Georges Pompidou, Assistance Publique-hopitaux De Paris, DOI 10.1007/s00467-015-3158-7 Pediatr Nephrol (2015) 30:1543–1730• OnabotulinumtoxinA is a safe and effective treatment for therapy resistant OAB in non-neuropathic children. • It can be a useful treatment option for therapy resistant incontinence and/or enuresis. • With one single treatment, over 50% cure rate may be achieved in a therapy resistant patient population. • The aim of this study is to analyze results and side effects after OnabotulinumtoxinA detrusor injection treatment in children in order to define its place in the treatment of non-neuropathic OAB • Effect on both incontinence and enuresis is reported. • Therapy resistant enuresis is to our knowledge not previously reported as indication for the use of OnabotulinumtoxinA RESULTS OF ONABOTULINUMTOXIN-A IN CHILDREN WITH THERAPY RESISTANT OVERACTIVE BLADDER: 10-YEAR EXPERIENCE

Studying nonobstructive azoospermia in cystinosis: histologic examination of testes and epididymis and sperm analysis in a Ctns−/− mouse model

OBJECTIVE To study the pathogenesis of male infertility in cystinosis due to nonobstructive azoospermia, using a Ctns(-/-) mouse model. DESIGN Observational case-control study. SETTING Academic research laboratory. ANIMAL(S) Male C57BL/6 Ctns(-/-) mice were compared with C57BL/6 wild-type (wt) mice. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Fertility was studied using litter size (n = 3 vs. n = 2). After animals were sacrificed, testes, epididymis, and vas deferens were removed for testicular cystine measurements (n = 5 vs. n = 6), histologic studies (n = 3 vs. n = 3), and sperm analysis (n = 3 vs. n = 3). RESULT(S) Mean testicular cystine content was significantly higher in Ctns(-/-) mice compared with wt mice (26.6 ± 1.22 vs. 0.1 ± 0.01 nmol cystine/mg protein). Testes of Ctns(-/-) mice had lower weight compared with wt mice (0.096 ± 0.009 g vs. 0.112 ± 0.004 g), but mice fertility was similar (litter size 6.6 ± 1.4 vs. 6.3 ± 2.6 pups). Neither histologic nor sperm abnormalities were found. CONCLUSION(S) The Ctns(-/-) mouse model generated on C57BL/6 background is not suitable for clarifying the pathogenesis of male infertility in cystinosis. The etiology of nonobstructive azoospermia in these patients remains unclear.